TRAF-3 gene products are signaling molecules that interact with the cytoplasmic tails of CD40 (Cheng et al., 1995; Hu et al., 1994; Sato et al., 1995), other Tumor Necrosis Factor-Receptor (TNF-R) family members (e.g. LTβ-R, CD30, CD27, Ox40) (Mosialos et al., 1995; Gedrich et al., 1996; Boucher et al., 1997; Yamamoto et al., 1998; Vanarsdale et al., 1997; Arch and Thompson, 1998; Kawamata et al., 1998) and the Epstein-Barr virus latent membrane protein, LMP1 (Mosialos et al., 1995). The finding that TRAF-3(−/−) lymphocytes are specifically defective in T-B lymphocyte collaboration, indiates that TRAF-3 gene products are required for signaling events that underlie this function (Xu et al., 1996). Full-length TRAF-3 alone is unlikely to account for such signaling, since over-expression of full-length TRAF-3 fails to induce NF-κB activation (Rothe et al., 1995; Takeuchi et al., 1996; Dadgostar and Cheng, 1998). However, alternative splicing of TRAF-3 transcripts generates mRNA species that encode at least 3 putative isoforms with altered Zn finger domains that may participate in transmitting receptor signals to the nucleus (Sato et al., 1995; Krajewski et al., 1997; van Eyndhoven et al., 1998). Therefore, the present study addressed whether TRAF-3 mRNA splice-deletion variants encode TRAF-3 protein isoforms that are able to induce NF-κB activation.
TRAF-3 is a member of the TRAF (TNF Receptor-associated factor) family of proteins, of which six have been identified (TRAF-1 through 6) (Rothe et al., 1994; Regnier et al., 1995; Ishida et al., 1996; Ishida et al., 1996; Kashiwada et al., 1998; Cheng et al., 1995; Hu et al., 1994; Sato et al., 1995; Mosialos et al., 1995). TRAF-3, like other TRAFs, appears to lack intrinsic catalytic activity, which suggests that TRAF-3 functions as a docking or adaptor molecule for other proteins that mediate signaling events. TRAF family members are related by significant homology in their carboxy-terminal TRAF-C domains (Rothe et al., 1994; Cheng et al., 1995). The TRAF-3 TRAF-C domain is known to be important for the interaction of TRAF-3 with the cytoplasmic tails of TNF-R family receptors (Cheng et al., 1995; Force et al., 1997; Vanarsdale et al., 1997), homo-oligomerization (Cheng et al., 1995; Force et al., 1997; Sato et al., 1995; Pullen et al., 1998) and binding to cytoplasmic proteins such as I-TRAF/TANK (Rothe et al., 1996; Cheng and Baltimore, 1996) and NIK (Song et al., 1997; Malinin et al., 1997). In addition to the TRAF-C domain, TRAF-3 contains an amino-terminal RING finger domain, five atypical Zn finger motifs, an iso-leucine zipper domain and a TRAF-N domain (Cheng et al., 1995; Hu et al., 1994; Sato et al., 1995; Mosialos et al., 1995). In TRAF-2 and TRAF-5, the RING finger and Zn finger domains have been shown to play important roles in mediating NF-κB activation (Takeuchi et al., 1996; Dadgostar and Cheng, 1998). The functional potentials of the TRAF-3 RING finger and Zn finger domains remain enigmatic, since TRAF-3 itself fails to induce NF-κB activation (Rothe et al., 1995; Takeuchi et al., 1996; Dadgostar and Cheng, 1998). However, the TRAF-3 RING finger domain is capable of supporting NF-κB activation in chimeric TRAF-3/5 molecules (Dadgostar and Cheng, 1998).
Alteration of the TRAF-3 Zn finger domain by alternative mRNA splicing was suggested by analysis of the sequences of the initial TRAF-3 cDNA clones isolated. One TRAF-3 cDNA clone (CAP1) contains a 75 bp deletion, relative to 3 other TRAF-3 cDNA clones (termed CRAF1, CD40 bp and LAP1) (Cheng et al., 1995; Hu et al., 1994; Sato et al., 1995; Mosialos et al., 1995). In addition, 2 other TRAF-3 mRNA variants containing 156 bp and 168 bp deletions were recently identified (van Eyndhoven et al., 1998). Each of the 3 mRNA species with deleted elements, encodes a putative TRAF-3 protein isoform with an altered number and composition of the Zn fingers. These putative isoforms have been termed, TRAF-3b (Δ25aa), TRAF-3c (Δ52aa) and TRAF-3d (Δ56aa) (Krajewski et al., 1997; van Eyndhoven et al., 1998). Characterization of the human TRAF-3 genomic structure indicated that these 3 TRAF-3 mRNA species result from alternative mRNA splicing (van Eyndhoven et al., 1998). Further analysis of the TRAF-3 gene suggested that a large number of additional splice variants may be generated, since each of the splice junctions of exons 4 through 10 are class “0”. Therefore, splice-deletion variants involving any or all of exons 5-10 would maintain open reading frames (ORFs) (van Eyndhoven et al., 1998). However, additional splice-deletion variants have not been characterized and the functions of the identified or predicted splice-deletion variants have not been studied.